During the last years there is an increasing body of evidence that detection and characterization of tumor cells in bone marrow or peripheral blood of breast cancer patients may be clinically relevant in terms of disease-free interval and overall survival (A. C. Lambrechts et al; 1998). Moreover, the prospective evaluation of minimal residual disease (MRD) may give information concerning the effectiveness of adjuvant therapy (K. Pantel et al; 2003). Therefore, highly sensitive methods for the early detection of circulating cancer cells are very important for the early diagnosis and more effective treatment of MRD.
The intermediate filament cytokeratin-19 (CK-19) is stably and abundantly expressed in the majority of epithelial tumor cells and is one of the most frequently used markers for the detection of occult tumor cells in the peripheral blood of patients with breast cancer (S. Braun et al; 2000; Y. H. Datta et al; 1994; A. Schoenfield et al; 1997). The present inventors have recently shown that the detection of CK-19 mRNA positive cells in the peripheral blood represents one of the most powerful determinants of outcome in patients with operable breast cancer before the initiation of any adjuvant treatment, with patients negative for CK-19 mRNA having a better chance of long-term survival and disease free interval (A. Stathopoulou et al; 2002).
Furthermore, in a previous study we have developed a quantitative method based on real-time monitoring during PCR of fluorescently-labeled specific hybridization probes for CK-19 mRNA (A. Stathopoulou et al; 2003). By applying that method in patients with breast cancer, either stage I/II (operable) or IV (metastatic), as well as, in healthy blood donors we have found positive cells in 70/337 (20.77%) and in 2/89 (2.2%) peripheral blood samples, respectively. In this way, we observed a false positive rate (2.2%) for normal blood donors, when a cutoff level of 0.6 MCF-7 cell equivalents/5 μg RNA (detection limit of the method) was set. By using this statistically calculated cut-off, some peripheral blood samples of patients and healthy donors were regarded as negative, despite showing an amplification curve for CK-19 at very high crossing points (Cps). These amplification curves were due to amplification of low level illegitimately transcribed CK-19 from hematopoietic cells (J. A. L pez-Guerrero et al; 1997), CK-19a and CK-19b pseudogenes (P. Ruud et al; 1999; E. S. Savtchenko et al; 1988) or amplification of contaminating genomic DNA, co extracted with total RNA from our samples. However, for samples found to be very close to this cut-off, the interpretation of this “grey zone” results was very critical for the treatment of our early breast cancer patients (V. Bozionellou et al; in press).
Thus there still exists a need for improved primers and methods for quantitative determination of mRNA transcripts in a biological sample. In particular there exists a need for improved primers and methods for determination of CK-19 mRNA positive cells in peripheral blood of operable cancer patients, which methods gives reduced background compared with previously known primers and methods, a high sensibility and a low frequency of false positives.